No description available.

Tropical seagrass ecosystems are highly productive and important for sustaining marine life and associated coastal societies. In this study, the diversity and role of nitrogen-fixing cyanobacteria associated with five common seagrass genera in coastal regions of the western Indian Ocean (WIO; Tanzania) were examined, as well as the impact of anthropogenic activities. Cyanobacteria were characterized morphologically and genetically (16S rRNA and nifH gene phylogeny), as were diel variations in nifH gene expression, NifH protein levels and nitrogenase activity. The results revealed that WIO seagrass beds supported rich cyanobacterial diversity and that these represented approx. 83% of total clones obtained (DNA and RNA nifH clone libraries). Non-heterocystous genera, such as Oscillatoria, Lyngbya, Leptolyngbya, Phormidium and Microcoleus dominated, while heterocystous morphotypes such as Calothrix were less frequent and unicellular morphotypes (e.g. Gloeocapsa, Chroococcus and Chroococcidiopsis) were few. Additionally, the phylogenetic analysis revealed several novel uncharacterized cyanobacterial clades. Cyanobacterial composition and nitrogenase activity varied over seasons and between the seagrass species. Day time nitrogenase activity originated primarily from heterocystous phylotypes, while non-heterocystous filamentous phylotypes fixed nitrogen at night. The highest activity in the diel cycle was 358 ± 232 nmol C2H4 g-1 h-1at 09.00 associated with epiphytes of the seagrass Cymodocea. Nitrogenase activity was consistently lower in anthropogenically disturbed (eutrophication) seagrass sites. Such data suggest that diazotrophic cyanobacteria may be a significant source of ‘new’ nitrogen in the often oligotrophic coastal regions of tropical oceans. It is also proposed that the rapid shifts in the cyanobacterial population and function found may also be used as early disturbance indicator in coastal management practices. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript. / SIDA SAREC Bilateral Marine Sciences Project / The Swedish Foundation for International Cooperation in Research and Higher Education

A large proportion of the world’s nitrogen needs is derived from symbiotic nitrogen fixation (SNF), which contributes substantially to agricultural sustainability. The partnership between legumes and rhizobia result in the formation of specialised structures called root nodules. Within these nodules SNF is supported by the sucrose transported from the leaves to the nodules for respiration. The end products of SNF in soybean (Glycine max (L.) Merr.) root nodules, namely ureides, are transported to the upper parts of the plant to supply nitrogen. Symbiotic nitrogen fixation provides a vital advantage for the production of soybean compared with most grain crops in that soybean fixes the nitrogen required for its growth and for the production of the high-protein content in seed and oil. The process of SNF is dramatically affected by drought, salt, cold and heavy metal stresses. Since SNF is such an important yield-determining factor, a lack in understanding these complexes inevitably delays progress towards the genetic improvement of soybean genotypes and also complicates decisions with regard to the suitability of certain genotypes for the various soybean producing areas in South Africa. The largest soybean producing areas in South Africa are situated at high altitudes, with minimum daily temperatures which can be critically low and impeding the production of soybean. Soybean is chilling sensitive, with growth, development and yield being affected negatively at temperatures below 15°C. Dark chilling (low night temperature) stress has proved to be one of the most important restraints to soybean production in South Africa. Among the symptoms documented in dark chilling sensitive soybean genotypes are reduced growth rates, loss of photosynthetic capacity and pigment content, as well as premature leaf senescence and severely inhibited SNF. Existing knowledge about stress-induced nodule senescence is based on fragmented information in the literature obtained in numerous, and often diverse, legume species. The precise nature and sequence of events participating in nodule senescence has not yet been fully explained. The main objectives of this investigation were to characterise the natural senescence process in soybean nodules under optimal growth conditions and to characterise the alteration of the key processes of SNF in a chilling sensitive soybean genotype during dark chilling. Moreover, to establish whether recovery in nodule functionality following a long term dark chilling period occured, or whether nodule senescence was triggered, and if sensitive biochemical markers of premature nodule senescence could be identified. A known chilling sensitive soybean genotype, PAN809, was grown under controlled growth conditions in a glasshouse. To determine the baseline and change over time for key parameters involved in SNF, a study was conducted under optimal growing conditions over a period of 6 weeks commencing 4 weeks after sowing. The cluster of crown nodules were monitored weekly and analysis included nitrogenase activity, ureide content, respiration rate, leghemoglobin content, sucrose synthase (SS) activity and sucrose content. Further investigations focused on induced dark chilling effects on nodule function to determine the alterations in key parameters of SNF. Plants were subjected to dark chilling (6˚C) for 12 consecutive nights and kept at normal day temperatures (26˚C). The induced dark chilling was either only shoot (SC) exposure or whole plant chilling (WPC). These treatments were selected since, in some areas in South Africa cold nights result not only in shoot chilling (SC) but also in low soil temperatures causing direct chilling of both roots and shoots. To determine if premature nodule senescence was triggered, the recovery following 12 consecutive nights of chilling treatment was monitored for another 4 weeks. It was established that the phase of optimum nitrogenase activity under optimal growing conditions occurred during 4 to 6 weeks after sowing where after a gradual decline commenced. This decline was associated with a decline in nitrogenase protein content and an increase in ureide content. The stability of SS activity and nodule respiration showed that carbon-dependent metabolic processes were stable for a longer period than previously mentioned parameters. The negative correlation that was observed between nitrogenase activity and nodule ureide content pointed towards the possible presence of a feedback inhibition trigger on nitrogenase activity. A direct effect of dark chilling on nitrogenase activity and nodule respiration rate led to a decline in nodule ureide content that occurred without any limitations on the carbon flux of the nodules (i.e. stable sucrose synthase activity and nodule sucrose content). The effect on SC plants was much less evident but did indicate that currently unknown shoot-derived factors could be involved in the minor inhibition of SNF. It was concluded that the repressed rates of respiration might have led to increased O2 concentrations in the nodule, thereby inhibiting the nitrogenase protein and so the production of ureides. It was found that long term chilling severely disrupted nitrogenase activity and ureide synthesis in nodules. Full recovery in all treatments occurred after 2 weeks of suspension of dark chilling, however, this only occurred when control nodules already commenced senescence. This points toward reversible activation of the nitrogenase protein with no evidence in support of premature nodule senescence. An increase in intercellular air space area was induced by long term dark chilling in nodules, specifically by the direct chilling of nodules (WPC treatment). The delayed diminishment of intercellular air space area back to control levels following dark chilling may be an important factor involved in the recovery of nitrogenase activity because enlarged air spaces would have favoured gaseous diffusion, and hence deactivation of nitrogenase, in an elevated O2 environment (due to supressed nodule respiration rates). These findings revealed that dark chilling did not close the diffusion barrier, as in the case of drought and other stress factors, but instead opened it due to an increase in air space areas in all regions of the nodule. In conclusion, this study established that dark chilling did not initiate premature nodule senescence and that SNF demonstrated resilience, with full recovery possible following even an extended dark chilling period involving low soil temperatures. / Thesis(PhD (Botany))--North-West University, Potchefstroom Campus, 2013

A large proportion of the world’s nitrogen needs is derived from symbiotic nitrogen fixation (SNF), which contributes substantially to agricultural sustainability. The partnership between legumes and rhizobia result in the formation of specialised structures called root nodules. Within these nodules SNF is supported by the sucrose transported from the leaves to the nodules for respiration. The end products of SNF in soybean (Glycine max (L.) Merr.) root nodules, namely ureides, are transported to the upper parts of the plant to supply nitrogen. Symbiotic nitrogen fixation provides a vital advantage for the production of soybean compared with most grain crops in that soybean fixes the nitrogen required for its growth and for the production of the high-protein content in seed and oil. The process of SNF is dramatically affected by drought, salt, cold and heavy metal stresses. Since SNF is such an important yield-determining factor, a lack in understanding these complexes inevitably delays progress towards the genetic improvement of soybean genotypes and also complicates decisions with regard to the suitability of certain genotypes for the various soybean producing areas in South Africa. The largest soybean producing areas in South Africa are situated at high altitudes, with minimum daily temperatures which can be critically low and impeding the production of soybean. Soybean is chilling sensitive, with growth, development and yield being affected negatively at temperatures below 15°C. Dark chilling (low night temperature) stress has proved to be one of the most important restraints to soybean production in South Africa. Among the symptoms documented in dark chilling sensitive soybean genotypes are reduced growth rates, loss of photosynthetic capacity and pigment content, as well as premature leaf senescence and severely inhibited SNF. Existing knowledge about stress-induced nodule senescence is based on fragmented information in the literature obtained in numerous, and often diverse, legume species. The precise nature and sequence of events participating in nodule senescence has not yet been fully explained. The main objectives of this investigation were to characterise the natural senescence process in soybean nodules under optimal growth conditions and to characterise the alteration of the key processes of SNF in a chilling sensitive soybean genotype during dark chilling. Moreover, to establish whether recovery in nodule functionality following a long term dark chilling period occured, or whether nodule senescence was triggered, and if sensitive biochemical markers of premature nodule senescence could be identified. A known chilling sensitive soybean genotype, PAN809, was grown under controlled growth conditions in a glasshouse. To determine the baseline and change over time for key parameters involved in SNF, a study was conducted under optimal growing conditions over a period of 6 weeks commencing 4 weeks after sowing. The cluster of crown nodules were monitored weekly and analysis included nitrogenase activity, ureide content, respiration rate, leghemoglobin content, sucrose synthase (SS) activity and sucrose content. Further investigations focused on induced dark chilling effects on nodule function to determine the alterations in key parameters of SNF. Plants were subjected to dark chilling (6˚C) for 12 consecutive nights and kept at normal day temperatures (26˚C). The induced dark chilling was either only shoot (SC) exposure or whole plant chilling (WPC). These treatments were selected since, in some areas in South Africa cold nights result not only in shoot chilling (SC) but also in low soil temperatures causing direct chilling of both roots and shoots. To determine if premature nodule senescence was triggered, the recovery following 12 consecutive nights of chilling treatment was monitored for another 4 weeks. It was established that the phase of optimum nitrogenase activity under optimal growing conditions occurred during 4 to 6 weeks after sowing where after a gradual decline commenced. This decline was associated with a decline in nitrogenase protein content and an increase in ureide content. The stability of SS activity and nodule respiration showed that carbon-dependent metabolic processes were stable for a longer period than previously mentioned parameters. The negative correlation that was observed between nitrogenase activity and nodule ureide content pointed towards the possible presence of a feedback inhibition trigger on nitrogenase activity. A direct effect of dark chilling on nitrogenase activity and nodule respiration rate led to a decline in nodule ureide content that occurred without any limitations on the carbon flux of the nodules (i.e. stable sucrose synthase activity and nodule sucrose content). The effect on SC plants was much less evident but did indicate that currently unknown shoot-derived factors could be involved in the minor inhibition of SNF. It was concluded that the repressed rates of respiration might have led to increased O2 concentrations in the nodule, thereby inhibiting the nitrogenase protein and so the production of ureides. It was found that long term chilling severely disrupted nitrogenase activity and ureide synthesis in nodules. Full recovery in all treatments occurred after 2 weeks of suspension of dark chilling, however, this only occurred when control nodules already commenced senescence. This points toward reversible activation of the nitrogenase protein with no evidence in support of premature nodule senescence. An increase in intercellular air space area was induced by long term dark chilling in nodules, specifically by the direct chilling of nodules (WPC treatment). The delayed diminishment of intercellular air space area back to control levels following dark chilling may be an important factor involved in the recovery of nitrogenase activity because enlarged air spaces would have favoured gaseous diffusion, and hence deactivation of nitrogenase, in an elevated O2 environment (due to supressed nodule respiration rates). These findings revealed that dark chilling did not close the diffusion barrier, as in the case of drought and other stress factors, but instead opened it due to an increase in air space areas in all regions of the nodule. In conclusion, this study established that dark chilling did not initiate premature nodule senescence and that SNF demonstrated resilience, with full recovery possible following even an extended dark chilling period involving low soil temperatures. / Thesis(PhD (Botany))--North-West University, Potchefstroom Campus, 2013

As cianobactérias caracterizam-se como o grupo de micro-organismos fotoautotrófico mais abundante encontrado nas regiões polares. Representantes deste grupo realizam a fotossíntese oxigênica e também podem fixar o nitrogênio atmosférico. A maioria dos levantamentos da comunidade de cianobactérias na Antártica tem sido realizada apenas por meio de observações microscópicas de amostras ambientais. O isolamento de linhagens e consequentes estudos fisiológicos, bem como, análises independentes de cultivo ainda são escassos. Neste estudo a comunidade de cianobactérias de duas ilhas oceânicas da Antártica foi investigada utilizando abordagens moleculares dependentes e independentes de cultivo. O papel ecológico das cianobactérias como fornecedoras de formas assimiláveis de nitrogênio e o potencial genético para biossíntese de produtos naturais também foi avaliado. Sessenta e oito linhagens de cianobactérias foram isoladas a partir de diferentes substratos coletados. Elas pertencem às ordens Chroococcales, Pseudanabaenales, Oscillatoriales e Nostocales, famílias Xenococcaceae, Dermocarpellaceae, Pseudanabaenaceae, Oscillatoriaceae, Nostocaceae, Microchaetaceae e Rivulariaceae. Análises filogenéticas baseadas nas sequências de RNAr 16S dessas cianobactérias revelou a existência de agrupamentos: formado exclusivamente por sequência de linhagem isolada nesse trabalho; composto por sequências antárticas oriundas desse e de outros trabalhos desenvolvidos em outras regiões antárticas; e por sequências originárias de diversas regiões do mundo. Quarenta e uma linhagens apresentaram fragmento do gene nifH, responsável pela codificação do complexo enzimático da nitrogenase, o qual está envolvido na fixação biológica do nitrogênio (FBN). Formas unicelulares (Chroococcales), homocitadas (Pseudanabaenales e Oscillatoriales) e heterocitadas (Nostocales) apresentaram potencial genético para realização da FBN, e 18 delas foram submetidas aos testes de redução de acetileno (ARA) com alta sensibilidade de detecção. Todas as linhagens testadas exibiram alguma atividade em resposta a diferentes concentrações de oxigênio e/ou a luminosidade em diferentes condições de temperatura. Filogeneticamente, as sequências do gene nifH apresentaram três padrões distintos de agrupamento, o que pode estar relacionado aos eventos evolutivos envolvidos na distribuição e ou manutenção deste gene. A presença de genes e ou regiões intergênicas evidenciaram o elevado potencial genético dessas linhagens para sintetizar produtos naturais com interesse biotecnológico. A abundância no número de cópias do gene nifH relacionado às cianobactérias nas amostras de biofilme reforça a importância desse grupo de microorganismos como fornecedor de formas reduzidas de N para o ambiente antártico. A análise da comunidade de cianobactérias por meio do sequenciamento do RNAr 16S de DNA metagenômico evidenciou predominância de UTOs relacionadas às ordens Nostocales, Oscillatoriales e Pseudanabaenales, famílias Pseudanabaenaceae, Phormidiaceae, Nostocaceae e Rivulariaceae. A árvore filogenética contendo as sequências de cianobactérias cultivadas e não-cultivadas mostrou que somente parte da comunidade presente em biofilmes foi acessada por isolamento, indicando a complementariedade entre as duas abordagens utilizadas na análise da comunidade de cianobactérias / Cyanobacteria are characterized as the most abundant group of photoautotrophic microorganisms found in the polar regions. Members of this group perform oxygenic photosynthesis and many of them can also fix atmospheric nitrogen. Investigations on the cyanobacterial community have been made mainly applying microscopic observations of environmental samples. Cyanobacterial isolation, physiological studies and cultureindependent analyses are scarce. In this study the cyanobacterial community from two oceanic islands in Antarctica was investigated using culture-dependent and independent approaches. Also, the ecological role of this group of microorganisms as nitrogen-fixing organisms and the genetic potential for biosynthesis of natural products were evaluated. Sixty-eight cyanobacterial strains were isolated from different environmental samples. They belong to the orders Chroococcales, Pseudanabaenales, Oscillatoriales and Nostocales, families Xenococcaceae, Dermocarpellaceae, Pseudanabaenaceae, Oscillatoriaceae, Nostocaceae, Microchaetaceae and Rivulariaceae. Phylogenetic analyses based on 16S rRNA sequences of these cyanobacteria revealed the existence of groups: exclusively formed by sequence of strain isolated in this work; intermixed sequences from this and other studies developed in other Antarctic regions; and sequences originated from different regions of the world. Fortyone cultured strains possess the nifH gene fragment encoding the nitrogenase enzyme complex, which is related to the biological nitrogen fixation (BNF). Unicellular (Chroococcales), homocytous (Pseudanabaenales and Oscillatoriales) and heterocytous forms (Nostocales) showed genetic potential for BNF, and 18 of them were subjected to acetylene reduction assay (ARA) coupled with a sensitive laser photoacoustic ethylene detector. All strains tested exhibited some nitrogenase activity in response to different concentrations of oxygen and or irradiance under different temperature conditions. Phylogenetically, the nifH gene sequences showed three distinct grouping patterns that may be related to the evolutionary events involved in the distribution and or maintenance of this gene. The presence of genes and or intergenic regions in these cyanobacterial strains underscores the genetic potential of them to synthesize natural products with biotechnological interest.The abundance of nifH gene copies related to cyanobacteria in biofilm samples highlights the importance of this group of microorganisms as suppliers of N reduced forms for Antarctic environment. The analysis of the cyanobacteria community revealed by 16S rRNA sequencing of metagenomic DNA showed a predominance of OTUs related to orders Nostocales, Oscillatoriales and Pseudanabaenales, families Pseudanabaenaceae, Phormidiaceae, Nostocaceae and Rivulariaceae. The phylogenetic tree containing Antarctic sequences from cultivated and uncultivated cyanobacteria showed that only part of this community in biofilms has been accessed by isolation, indicating the complementarity between the two approaches used in the analysis of cyanobacterial community

As cianobactérias caracterizam-se como o grupo de micro-organismos fotoautotrófico mais abundante encontrado nas regiões polares. Representantes deste grupo realizam a fotossíntese oxigênica e também podem fixar o nitrogênio atmosférico. A maioria dos levantamentos da comunidade de cianobactérias na Antártica tem sido realizada apenas por meio de observações microscópicas de amostras ambientais. O isolamento de linhagens e consequentes estudos fisiológicos, bem como, análises independentes de cultivo ainda são escassos. Neste estudo a comunidade de cianobactérias de duas ilhas oceânicas da Antártica foi investigada utilizando abordagens moleculares dependentes e independentes de cultivo. O papel ecológico das cianobactérias como fornecedoras de formas assimiláveis de nitrogênio e o potencial genético para biossíntese de produtos naturais também foi avaliado. Sessenta e oito linhagens de cianobactérias foram isoladas a partir de diferentes substratos coletados. Elas pertencem às ordens Chroococcales, Pseudanabaenales, Oscillatoriales e Nostocales, famílias Xenococcaceae, Dermocarpellaceae, Pseudanabaenaceae, Oscillatoriaceae, Nostocaceae, Microchaetaceae e Rivulariaceae. Análises filogenéticas baseadas nas sequências de RNAr 16S dessas cianobactérias revelou a existência de agrupamentos: formado exclusivamente por sequência de linhagem isolada nesse trabalho; composto por sequências antárticas oriundas desse e de outros trabalhos desenvolvidos em outras regiões antárticas; e por sequências originárias de diversas regiões do mundo. Quarenta e uma linhagens apresentaram fragmento do gene nifH, responsável pela codificação do complexo enzimático da nitrogenase, o qual está envolvido na fixação biológica do nitrogênio (FBN). Formas unicelulares (Chroococcales), homocitadas (Pseudanabaenales e Oscillatoriales) e heterocitadas (Nostocales) apresentaram potencial genético para realização da FBN, e 18 delas foram submetidas aos testes de redução de acetileno (ARA) com alta sensibilidade de detecção. Todas as linhagens testadas exibiram alguma atividade em resposta a diferentes concentrações de oxigênio e/ou a luminosidade em diferentes condições de temperatura. Filogeneticamente, as sequências do gene nifH apresentaram três padrões distintos de agrupamento, o que pode estar relacionado aos eventos evolutivos envolvidos na distribuição e ou manutenção deste gene. A presença de genes e ou regiões intergênicas evidenciaram o elevado potencial genético dessas linhagens para sintetizar produtos naturais com interesse biotecnológico. A abundância no número de cópias do gene nifH relacionado às cianobactérias nas amostras de biofilme reforça a importância desse grupo de microorganismos como fornecedor de formas reduzidas de N para o ambiente antártico. A análise da comunidade de cianobactérias por meio do sequenciamento do RNAr 16S de DNA metagenômico evidenciou predominância de UTOs relacionadas às ordens Nostocales, Oscillatoriales e Pseudanabaenales, famílias Pseudanabaenaceae, Phormidiaceae, Nostocaceae e Rivulariaceae. A árvore filogenética contendo as sequências de cianobactérias cultivadas e não-cultivadas mostrou que somente parte da comunidade presente em biofilmes foi acessada por isolamento, indicando a complementariedade entre as duas abordagens utilizadas na análise da comunidade de cianobactérias / Cyanobacteria are characterized as the most abundant group of photoautotrophic microorganisms found in the polar regions. Members of this group perform oxygenic photosynthesis and many of them can also fix atmospheric nitrogen. Investigations on the cyanobacterial community have been made mainly applying microscopic observations of environmental samples. Cyanobacterial isolation, physiological studies and cultureindependent analyses are scarce. In this study the cyanobacterial community from two oceanic islands in Antarctica was investigated using culture-dependent and independent approaches. Also, the ecological role of this group of microorganisms as nitrogen-fixing organisms and the genetic potential for biosynthesis of natural products were evaluated. Sixty-eight cyanobacterial strains were isolated from different environmental samples. They belong to the orders Chroococcales, Pseudanabaenales, Oscillatoriales and Nostocales, families Xenococcaceae, Dermocarpellaceae, Pseudanabaenaceae, Oscillatoriaceae, Nostocaceae, Microchaetaceae and Rivulariaceae. Phylogenetic analyses based on 16S rRNA sequences of these cyanobacteria revealed the existence of groups: exclusively formed by sequence of strain isolated in this work; intermixed sequences from this and other studies developed in other Antarctic regions; and sequences originated from different regions of the world. Fortyone cultured strains possess the nifH gene fragment encoding the nitrogenase enzyme complex, which is related to the biological nitrogen fixation (BNF). Unicellular (Chroococcales), homocytous (Pseudanabaenales and Oscillatoriales) and heterocytous forms (Nostocales) showed genetic potential for BNF, and 18 of them were subjected to acetylene reduction assay (ARA) coupled with a sensitive laser photoacoustic ethylene detector. All strains tested exhibited some nitrogenase activity in response to different concentrations of oxygen and or irradiance under different temperature conditions. Phylogenetically, the nifH gene sequences showed three distinct grouping patterns that may be related to the evolutionary events involved in the distribution and or maintenance of this gene. The presence of genes and or intergenic regions in these cyanobacterial strains underscores the genetic potential of them to synthesize natural products with biotechnological interest.The abundance of nifH gene copies related to cyanobacteria in biofilm samples highlights the importance of this group of microorganisms as suppliers of N reduced forms for Antarctic environment. The analysis of the cyanobacteria community revealed by 16S rRNA sequencing of metagenomic DNA showed a predominance of OTUs related to orders Nostocales, Oscillatoriales and Pseudanabaenales, families Pseudanabaenaceae, Phormidiaceae, Nostocaceae and Rivulariaceae. The phylogenetic tree containing Antarctic sequences from cultivated and uncultivated cyanobacteria showed that only part of this community in biofilms has been accessed by isolation, indicating the complementarity between the two approaches used in the analysis of cyanobacterial community